Gut microbiota has recently emerged as a novel, metabolically active 'organ', tightly linked to obesity and related metabolic disorders such as diabetes and cardiovascular disease; and compelling evidence indicates that several key metabolites (TMAO, L-carnitine, bile acids subtypes) related to gut microbiota metabolism may play pivotal roles in mediating the metabolic effects. Diet is a major force that shapes gut microbiota and weight loss may alter metabolism of gut microbiota. The goal of this application is to examine the long-term (2 years) effects of weight-loss diets varying in macronutrients (fat, protein, and carbohydrate) on the novel cardiometabolic metabolites involved in gut microbiota metabolism in 811 overweight or obese patients from thus far one of the largest and most comprehensive comparator trials on the popular weight-loss diets - the POUNDS LOST trial. We will also assess the relation of changes in plasma gut microbiota metabolites with changes in markers of energy intake and expenditure, including gut hormones (GLP-1, ghrelin), resting metabolic rate (RMR), and total energy expenditure (TEE) measured by doubly-labeled water (DLW); as well as with changes in metabolic risk factors including insulin sensitivity, lipids, adipokines, inflammation, and amino acids. In addition, we will examine the associations between gut microbiota metabolites and global adipose gene expression. Because measurement of energy balance and metabolic markers, and adipose gene expression are funded through separate grants, the proposed project will be conducted in an extremely cost-efficient manner. We have assembled a solid group of experienced collaborators with expertise in epidemiology, metabolomics, gene expression, statistics, clinical trial, and nutrition. We believe that findings of this project will provide novel insights into the metabolic effects of gut microbiota and their relations with dietary factors; and considerably contribute to advance toward new prevention and treatment strategies for obesity and related metabolic disorders through diet intervention.